ABSTRACT
Quantitative real-time PCR (qRT-PCR), used to determine the gene expression profile, is an important tool in functional genomic research, including fishes. To obtain more robust and meaningful result, the best possible normalization of the data is of utmost significance. In the present study, we have evaluated the potential of five commonly used housekeeping genes i.e., elongation factor 1-α (EF1A), β-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-2-Microglobulin (B2M) in normal physiological conditions, developmental stages and in response to bacterial infection in Asian seabass, Lates calcarifer (Bloch), an important food fish cultured in the Asia-Pacific region. The expression levels of these five genes were estimated in 11 tissues of normal seabass juveniles, 14 embryonic and larval developmental stages and six tissues of Vibrio alginolyticus-challenged animals. Further, the expression stability of these genes was calculated based on three algorithms i.e. geNorm, NormFinder and BestKeeper. The results showed that although there are tissue-specific variations for each gene, ACTB and EF1A are the most stable genes across the tissues of normal animals. However, in bacteria-challenged animals, EF1A and 18S were found to be the best reference genes for data normalization. The expression of all the genes tested showed an increasing trend in developmental stages and the increase was significant at blastula stage. Among the five genes tested, EF1A and ACTB were found to be the genes with least variation and highest stability across the developmental stages. This forms the first report on validation of housekeeping genes in L. calcarifer, in the context of ontogenic development and in response to infection.
ABSTRACT
The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K+-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35‰ salinities and maintained for 20 days to observe long term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5‰ compared to 25‰ (isosmotic salinity) and 0.48 folds lower at 35‰. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5‰ the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25‰ and 35‰ NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25‰. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5‰ measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.